Randomly broken fragment PCR with 5′ end-directed adaptor for genome walking

Abstract

Many genome walking methods based on polymerase chain reaction (PCR) are available, including those with and without restriction enzyme modification. Nevertheless, these methods suffer from low reproducibility, inefficiency, and non-specificity. Here, we present a traceable and efficient PCR strategy: randomly broken fragment PCR with 5′ end-directed adaptor for genome walking. The genome is first fragmented randomly. After blunting ends, the fragments are ligated to the 5′ end-directed adaptors. Semi-nested PCR is then performed. Thus, we can obtain an unknown sequence by cloning the fragments of interest, followed by sequencing. This method effectively bypasses the above-mentioned obstacles and offers the advances: 1) genome fragmentation without using restriction enzymes; 2) enhancement of primer specificity and the prevention of self-ligation between the adaptors by employing a 5′ end-directed adaptor. All of the steps in this new method are straightforward, and the unknown sequence can be definitively obtained by merely applying the method once.

Avaliação de uma região <i>hotspot</i> do gene citocromo b para resistência aos fungicidas inibidores da quinona oxidase (QoI) em patógenos de uva Niágara Rosada

“…Nesse estudo, através da técnica de GW foi possível sequenciar 65% do gene cytB de um isolado brasileiro de P. viticola, não sendo observada a presença de íntrons; esse resultado é condizente com o que é descrito na literatura para essa espécie (GIRESSE et al, 2010;GRASSO et al, 2006). Apesar do gene cytB já ter sido sequenciado na espécie P. viticola, esse tipo de estudo ainda é importante pois já foi relatado que isolados de uma mesma espécie podem apresentar diferentes números e tamanhos de íntron, como descrito para Botrytis cinerea (LEROUX et al, 2010) e Saccharomyces cerevisiae (FOURY et al, 1998 (TAN et al, 2005;XU et al, 2013).…”

Next generation DNA sequencing based strategies; towards a new era for the traceability of endangered species and genetically modified organisms

“…DNA enrichment approaches a) Use of specific primers followed by universal primers Long template-Rapid Amplification of Genomic DNA Ends (LT-RADE) [228] Linear amplification-mediated PCR (LAM-PCR) [229] Non-restrictive Linear amplification-mediated PCR (nrLAM-PCR) [225] b) Use of semi-random primers followed by specific primers SiteFinding-PCR [227] c) Use of specific primer or semi-random and adapter primer simultaneously Locus-finding PCR (LF PCR) [230] High-throughput insertion tracking by deep sequencing (HITS) [224] Randomly broken fragment PCR (RBF-PCR) [231] sA-T linker adapter PCR [232] Loop-linker PCR [232] Approaches that do not use DNA pre-treatment, require first the synthesis of a single stranded target molecule, and are hence referred to as primer extension based approaches. Primer extension can be performed in a target gene specific or semi-random way.…”

“…Both enzyme and physical fragmentation approaches require subsequent end repair procedures (tailing or linker or adapter ligation) prior to either specific PCR or direct sequencing. Looplinker PCR [233], A-T linker [232], HITS [224], and RBF-PCR [231] come under one of the fragmentation approaches. Apart from the described approaches in the present paper, inverse PCR (I-PCR) [234,235], thermal asymmetric interlaced-PCR (TAIL-PCR) [236][237][238][239][240], ligation-mediated PCR (LM-PCR) [241], randomly primed PCR (RP-PCR) [236,242], vectorette PCR [243], boomerang PCR [244], TOPO vector ligation PCR [245], anchored PCR [246], cassette PCR [247] and T-Linker PCR [248] are some of the old enrichment approaches that were developed in the last two decades [39].…”

“…RBF-PCR approach ( Figure 5.9) was successfully performed to identify the unknown sequence adjacent to known sequence motifs in the GMO maize LY038 [231]. In this approach, the genomic DNA was randomly fragmented by ultrasonication and repaired by addition of adenines to the 3' end of the DNAstrands.…”

“…Instead, by adding a primer adapter primer (AP) that has the same sequence as one of the non-complementary parts of the adaptor, PCR can only occur when the complementary strand is synthesized in the first round of PCR by elongation of the target sequence specific primer (SP1). Subsequent nested PCR was performed to increase the specificity ( Figure 5.9) [231]. The RBF-PCR approach was performed with some modifications compared to HITS: a) the specific biotinylated primer was replaced by a specific primer (SP) and b) instead of Illumina NGS adapters, specifically designed APs used to avoid self-ligation ( Figure 5.9).…”